Field flow fractionation is a versatile technique for the high resolution separation of a wide variety of particulates suspended in a fluid medium. The particulates include macromolecules, generally in the 10.sup.5 to the 10.sup.13 molecular weight (0.001 to 1 .mu.m) range, colloids, particles, micelles, organelles and the like. The technique is more explicitly described in U.S. Pat. No. 3,449,938, issued June 17, 1969 to John C. Giddings and U.S. Pat. No. 3,523,610, issued Aug. 11, 1970 to Edward M. Purcell and Howard C. Berg.
Field flow fractionation is the result of the differential migration rate of components in a carrier or mobile phase in a manner similar to that of chromatography. However, in field flow fractionation there is no separate stationary phase as is in the case of chromatography. Sample retention is caused by the redistribution of sample components between the fast to the slow moving strata within the mobile phase. Thus, particulates elutes more slowly than the solvent front. Typically, a field flow fractionation channel, consisting of two closely spaced parallel surfaces, is used. A mobile phase is caused to flow continuously through the gap between the surfaces. Because of the narrowness of this gap or channel (typically 0.025 centimeters (cm)) the mobile phase flow is laminar with a characteristic parabolic velocity profile. The flow velocity is the highest at the middle of the channel and the lowest near the two channel surfaces.
An external force field of some type (the force fields include gravitational, thermal, electrical, fluid cross-flow and others as described variously by Giddings and Berg and Purcell), is applied transversely (perpendicular) to the channel surfaces or walls. This force field pushes the sample components in the direction of the slower moving strata near the outer wall. The buildup of sample concentration near the wall, however, is resisted by the normal diffusion of the particulates in a direction opposite to the force field. This results in a dynamic layer of component particles, each component with an exponential--concentration profile. The extent of retention is determined by the time-average position of the particulates within the concentration profile which is a function of the balance between the applied field strength and the opposing tendency of particles to diffuse.
In sedimentation field flow fractionation, use is made of a centrifuge to establish the force field required for the separation. For this purpose a long, thin, annular belt-like channel is made to rotate within a centrifuge about its own axis. The resultant centrifugal force causes components of higher density than the mobile phase to settle toward the outer wall of the channel. For equal particle density, because of their higher diffusion rate, smaller particulates will accumulate into a thicker layer against the outer wall than will larger particles. On the average, therefore, larger particulates are forced closer to the outer wall.
If now the fluid medium, which may be termed a mobile phase or solvent, is fed continuously in one end of the channel, it carries the sample components through the channel for later detection at the outlet of the channel. Because of the shape of the laminar velocity profile within the channel and the placement of particulates in that profile, solvent flow causes smaller particulates to elute first, followed by a continuous elution of sample components in the order of ascending particulate mass.
This elution of components, whether it be in sedimentation field flow fractionation or other forms of field flow fractionation or other forms of field flow fractionation, is subject to a problem known as band broadening. By "band broadening" is meant that phenomena which causes the various eluted components to be spread over a longer period of time or a larger mobile phase volume than would be desirable in order to form a sharp elution peak or elution profile. With broader bands, the separation between components tends to degrade, with peaks merging into each other. This decreases the ability to obtain accurate measurements as the peaks or to collect pure sample components. Band broadening is due to many factors which will be described hereinafter. Suffice it to say for the moment that band broadening degrades the quality of the separations and hence is an undesirable trait or characteristic of the separation technique that requires correction.